Compositions and methods for sample processing

Abstract

This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.

What is claimed is: 
     
         1 . A method for processing template nucleic acid molecules, comprising:
 (a) providing a plurality of partitions comprising (i) a plurality of template nucleic acid molecules and (ii) a plurality of barcode nucleic acid molecules coupled to a plurality of supports, wherein at least a subset of said plurality of barcode nucleic acid molecules comprise barcode sequences having barcode segments that are identical and barcode segments that are different;   (b) in said plurality of partitions, using barcode nucleic acid molecules of said plurality of barcode nucleic acid molecules and template nucleic acid molecules of said plurality of template nucleic acid molecules to synthesize a plurality of barcoded nucleic acid molecules comprising said barcode sequences or complements thereof; and   (c) recovering said plurality of barcoded nucleic acid molecules or derivatives thereof from said partitions.   
     
     
         2 . The method of  claim 1 , wherein said at least said subset of said plurality of barcode nucleic acid molecules comprise barcode sequences or complements thereof that are different across said plurality of partitions. 
     
     
         3 . The method of  claim 1 , wherein a given partition of said plurality of partitions comprises a single support of said plurality of supports, which single support comprises a subset of said plurality of barcode nucleic acid molecules comprising identical barcode sequences. 
     
     
         4 . The method of  claim 3 , wherein a support of said plurality of supports of an additional given partition of said plurality of partitions comprises an additional subset of said plurality of barcode nucleic acid molecules comprising identical barcode sequences, wherein said barcode sequences of said given partition comprise barcode segments that are different from barcode segments of said barcode sequences of said additional given partition. 
     
     
         5 . The method of  claim 4 , wherein said subset of said plurality of barcode nucleic acid molecules of said given partition comprises different nucleic acid sequences. 
     
     
         6 . The method of  claim 1 , wherein said barcode sequences comprise between 4 and 20 nucleotides. 
     
     
         7 . The method of  claim 1 , wherein said at least said subset of said plurality of barcode nucleic acid molecules comprise priming sequences. 
     
     
         8 . The method of  claim 7 , wherein said priming sequences are random N-mer sequences. 
     
     
         9 . The method of  claim 7 , wherein said priming sequences are targeted primer sequences. 
     
     
         10 . The method of  claim 7 , wherein said plurality of barcoded nucleic acid molecules or derivatives thereof comprise said priming sequences. 
     
     
         11 . The method of  claim 1 , wherein said at least said subset of said plurality of barcode nucleic acid molecules comprise unique molecular identifier sequences. 
     
     
         12 . The method of  claim 1 , wherein said plurality of supports is a plurality of beads. 
     
     
         13 . The method of  claim 12 , wherein said plurality of beads is a plurality of gel beads. 
     
     
         14 . The method of  claim 13 , wherein said plurality of gel beads is dissolvable or disruptable. 
     
     
         15 . The method of  claim 12 , wherein said plurality of barcode nucleic acid molecules are releasably coupled to said plurality of beads. 
     
     
         16 . The method of  claim 1 , wherein a barcode nucleic acid molecule of said plurality of barcode nucleic acid molecules is releasable from a support of said plurality of supports through cleavage of a linkage between said barcode nucleic acid molecule and said support. 
     
     
         17 . The method of  claim 1 , wherein a barcode nucleic acid molecule of said plurality of barcode nucleic acid molecules is covalently attached to a support of said plurality of supports via a moiety comprising a labile bond. 
     
     
         18 . The method of  claim 17 , wherein said labile bond is selected from the group consisting of a thermally cleavable bond, a disulfide bond, and an ultraviolet-sensitive bond. 
     
     
         19 . The method of  claim 17 , wherein said labile bond comprises an ester linkage, a vicinal diol linkage, a Diels-Alder linkage, a sulfone linkage, a silyl ester linkage, a glycosidic linkage, a peptide linkage, or a phosphodiester linkage. 
     
     
         20 . The method of  claim 1 , wherein said plurality of partitions comprises a plurality of droplets. 
     
     
         21 . The method of  claim 1 , wherein said plurality of partitions comprises a plurality of wells. 
     
     
         22 . The method of  claim 1 , wherein (c) comprises pooling contents of said plurality of partitions. 
     
     
         23 . The method of  claim 1 , wherein said plurality of template nucleic acid molecules is a plurality of ribonucleic acid molecules. 
     
     
         24 . The method of  claim 1 , further comprising determining sequences of barcoded nucleic acid molecules of said plurality of barcoded nucleic acid molecules. 
     
     
         25 . The method of  claim 24 , wherein said sequences of said barcoded nucleic acid molecules of said plurality of barcoded nucleic acid molecules identify said barcoded nucleic acid molecules as deriving from template nucleic acid molecules of said plurality of template nucleic acid molecules. 
     
     
         26 . The method of  claim 1 , wherein said plurality of barcoded nucleic acid molecules or derivatives thereof comprise sequences that are complementary to sequences of said template nucleic acid molecules. 
     
     
         27 . The method of  claim 1 , wherein (b) comprises annealing barcode nucleic acid molecules of said plurality of barcode nucleic acid molecules to template nucleic acid molecules of said plurality of template nucleic acid molecules and extending said barcode nucleic acid molecules annealed to said template nucleic acid molecules to generate barcoded nucleic acid molecules of said plurality of barcoded nucleic acid molecules. 
     
     
         28 . The method of  claim 27 , wherein (b) further comprises annealing barcode nucleic acid molecules of said plurality of barcode nucleic acid molecules to a subset of said barcoded nucleic acid molecules of said plurality of barcoded nucleic acid molecules and extending said barcode nucleic acid molecules annealed to said subset of said barcoded nucleic acid molecules to generate additional barcoded nucleic acid molecules. 
     
     
         29 . The method of  claim 1 , wherein in (b), a same template nucleic acid molecule of said plurality of template nucleic acid molecules is used to synthesize multiple barcoded nucleic acid molecules of said plurality of barcoded nucleic acid molecules. 
     
     
         30 . The method of  claim 1 , wherein said plurality of template nucleic acid molecules is derived from a plurality of cells.